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1.
Braz. j. oral sci ; 20: e219342, jan.-dez. 2021. ilus
Artigo em Inglês | LILACS, BBO - Odontologia | ID: biblio-1253927

RESUMO

Aim: The aim of this study was to compare the microtensile bond strength (µTBS) and the characteristics of the adhesive interface of Scotchbond Universal - SU ­ etch-and-rise mode (3M ESPE) and Adper Scotchbond Multi-Purpose - MP (3M ESPE) to dentin over time. Methods: Class I cavity preparations were performed in 60 human molars that were randomly divided according to the dentin bonding system (DBS) used (n=30): (1) Acid conditioning + SU and (2) Acid conditioning + MP. For bonding strength (BS) analysis, 30 teeth (n = 15) were sectioned into sticks and submitted to the microtensile test in a universal testing machine after 24 hours and 12 months. The adhesive interface of the others 30 teeth was analyzed in a confocal microscope after 24 hours and 12 months. The data of µTBS were analyzed by two-way repeated measures ANOVA and Tukey's HSD (α = 0.05). Results: SU presented the lowest DBS compared to MP (p=0.000). Time did not influenced DBS for both adhesive systems (p=0.177). Confocal microscopy analysis showed no cracks between both adhesive systems tested. Conclusion: The results indicate that MP - µTBS showed a better performance compared to SU in total-etch mode


Assuntos
Humanos , Adesivos Dentinários , Microscopia Confocal , Dentina , Metacrilatos
2.
Braz. j. oral sci ; 20: e211194, jan.-dez. 2021. ilus
Artigo em Inglês | LILACS, BBO - Odontologia | ID: biblio-1253930

RESUMO

Aim: The aim of this study was to evaluate the effect of ethanol-conditioned dentin on endodontic sealer penetration into dentinal tubules by confocal laser scanning microscopy (CLSM). Methods: Forty human maxillary anterior teeth were instrumented and divided into four groups (n = 10) according to the drying methods: 1) wet: vacuum only, 2) paper points: vacuum + absorbent paper points, (3) 70% ethanol: 70% ethanol (1 min) + vacuum + absorbent paper points, and (4) 100% ethanol: 100% ethanol (1 min) + vacuum + absorbent paper points. All root canals were filled with resin-based endodontic sealer. Four sections from each third (cervical, middle, and apical) were examined by CLSM. Root canal wall perimeter infiltrated by sealer, maximum depth of sealer penetration, percentage of penetrated area, and fluorescence intensity of rhodamine B were measured. Statistical analysis was performed by analysis of variance and Tukey's tests (α = 0.05). Results: No statistical difference was found when percentage of root canal wall coverage infiltrated by sealer were compared. The groups in which ethanol solutions were used presented greater depth of sealer penetration, higher percentage of penetrated area, and higher fluorescence intensity of rhodamine B (p< 0.05) when compared with the wet and paper point groups. Overall, 100% ethanol produced better results than 70% ethanol, except for rhodamine B intensity (cervical third). In addition, the absorbent paper points drying method behaved better than did vacuum only group, except for rhodamine B intensity (apical third). Conclusion: Ethanol-conditioned dentin improved the penetration of resin-based sealer into dentinal tubules, especially at the concentration of 100%


Assuntos
Humanos , Molhabilidade , Microscopia Confocal , Cimentos de Resina , Dentina , Etanol , Endodontia
3.
Molecules ; 26(13)2021 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-34209621

RESUMO

Silica nanoparticles (SiO2 NPs) synthesized by the Stober method were used as drug delivery vehicles. Doxorubicin hydrochloride (DOX·HCl) is a chemo-drug absorbed onto the SiO2 NPs surfaces. The DOX·HCl loading onto and release from the SiO2 NPs was monitored via UV-VIS and fluorescence spectra. Alternatively, the zeta potential was also used to monitor and evaluate the DOX·HCl loading process. The results showed that nearly 98% of DOX·HCl was effectively loaded onto the SiO2 NPs' surfaces by electrostatic interaction. The pH-dependence of the process wherein DOX·HCl release out of DOX·HCl-SiO2 NPs was investigated as well. For comparison, both the free DOX·HCl molecules and DOX·HCl-SiO2 NPs were used as the labels for cultured cancer cells. Confocal laser scanning microscopy images showed that the DOX·HCl-SiO2 NPs were better delivered to cancer cells which are more acidic than healthy cells. We propose that engineered DOX·HCl-SiO2 systems are good candidates for drug delivery and clinical applications.


Assuntos
Antineoplásicos , Doxorrubicina , Portadores de Fármacos , Nanopartículas , Neoplasias , Dióxido de Silício , Antineoplásicos/química , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Doxorrubicina/química , Doxorrubicina/farmacocinética , Doxorrubicina/farmacologia , Portadores de Fármacos/química , Portadores de Fármacos/farmacocinética , Portadores de Fármacos/farmacologia , Humanos , Células MCF-7 , Microscopia Confocal , Nanopartículas/química , Nanopartículas/uso terapêutico , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Neoplasias/patologia , Dióxido de Silício/química , Dióxido de Silício/farmacocinética , Dióxido de Silício/farmacologia
4.
Int J Mol Sci ; 22(12)2021 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-34203791

RESUMO

For in vitro modeling of human joints, osteochondral explants represent an acceptable compromise between conventional cell culture and animal models. However, the scarcity of native human joint tissue poses a challenge for experiments requiring high numbers of samples and makes the method rather unsuitable for toxicity analyses and dosing studies. To scale their application, we developed a novel method that allows the preparation of up to 100 explant cultures from a single human sample with a simple setup. Explants were cultured for 21 days, stimulated with TNF-α or TGF-ß3, and analyzed for cell viability, gene expression and histological changes. Tissue cell viability remained stable at >90% for three weeks. Proteoglycan levels and gene expression of COL2A1, ACAN and COMP were maintained for 14 days before decreasing. TNF-α and TGF-ß3 caused dose-dependent changes in cartilage marker gene expression as early as 7 days. Histologically, cultures under TNF-α stimulation showed a 32% reduction in proteoglycans, detachment of collagen fibers and cell swelling after 7 days. In conclusion, thin osteochondral slice cultures behaved analogously to conventional punch explants despite cell stress exerted during fabrication. In pharmacological testing, both the shorter diffusion distance and the lack of need for serum in the culture suggest a positive effect on sensitivity. The ease of fabrication and the scalability of the sample number make this manufacturing method a promising platform for large-scale preclinical testing in joint research.


Assuntos
Osso e Ossos/fisiologia , Custos e Análise de Custo , Técnicas de Cultura de Tecidos/economia , Técnicas de Cultura de Tecidos/métodos , Idoso , Idoso de 80 Anos ou mais , Agrecanas/genética , Agrecanas/metabolismo , Biomarcadores/metabolismo , Cartilagem Articular/metabolismo , Proliferação de Células , Sobrevivência Celular , Condrócitos/citologia , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Matriz Extracelular/metabolismo , Feminino , Humanos , Antígeno Ki-67/metabolismo , Masculino , Microscopia Confocal , Pessoa de Meia-Idade , Esclerose , Sobrevivência de Tecidos , Transcrição Genética , Fator de Necrose Tumoral alfa/metabolismo
5.
Int J Mol Sci ; 22(13)2021 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-34281255

RESUMO

Midazolam (MDZ) could affect lymphocyte immune functions. However, the influence of MDZ on cell's K+ currents has never been investigated. Thus, in the present study, the effects of MDZ on Jurkat T lymphocytes were studied using the patch-clamp technique. Results showed that MDZ suppressed the amplitude of delayed-rectifier K+ current (IK(DR)) in concentration-, time-, and state-dependent manners. The IC50 for MDZ-mediated reduction of IK(DR) density was 5.87 µM. Increasing MDZ concentration raised the rate of current-density inactivation and its inhibitory action on IK(DR) density was estimated with a dissociation constant of 5.14 µM. In addition, the inactivation curve of IK(DR) associated with MDZ was shifted to a hyperpolarized potential with no change on the slope factor. MDZ-induced inhibition of IK(DR) was not reversed by flumazenil. In addition, the activity of intermediate-conductance Ca2+-activated K+ (IKCa) channels was suppressed by MDZ. Furthermore, inhibition by MDZ on both IK(DR) and IKCa-channel activity appeared to be independent from GABAA receptors and affected immune-regulating cytokine expression in LPS/PMA-treated human T lymphocytes. In conclusion, MDZ suppressed current density of IK(DR) in concentration-, time-, and state-dependent manners in Jurkat T-lymphocytes and affected immune-regulating cytokine expression in LPS/PMA-treated human T lymphocytes.


Assuntos
Canais de Potássio de Retificação Tardia/antagonistas & inibidores , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/antagonistas & inibidores , Midazolam/farmacologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Animais , Citocinas/metabolismo , Canais de Potássio de Retificação Tardia/metabolismo , Relação Dose-Resposta a Droga , Flumazenil/farmacologia , Antagonistas de Receptores de GABA-A/farmacologia , Humanos , Hipnóticos e Sedativos/administração & dosagem , Hipnóticos e Sedativos/farmacologia , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/metabolismo , Células Jurkat , Cinética , Lipopolissacarídeos/farmacologia , Ativação Linfocitária , Microscopia Confocal , Midazolam/administração & dosagem , Técnicas de Patch-Clamp , Fito-Hemaglutininas/farmacologia , Linfócitos T/imunologia
6.
BMC Ophthalmol ; 21(1): 285, 2021 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-34301210

RESUMO

BACKGROUND: Corneal dystrophies are a group of rare, inherited disorders that are usually bilateral, symmetric, slowly progressive, and not related to environmental or systemic factors. The majority of publications present the advanced form of the disease with a typical clinical demonstration. The initial signs and symptoms of different epithelial and stromal corneal dystrophies are not specific; therefore, it is very important to establish the early characteristic corneal features of these disorders that could guide the diagnostic process. CASE PRESENTATION: The main purpose of this study was to report the differential diagnosis of a pediatric patient with bilateral anterior corneal involvement suspected of corneal dystrophy. An 8-year-old male patient presented with asymptomatic, persistent, superficial, bilateral, diffuse, anterior corneal opacities. Slit lamp examination results were not specific. Despite the lack of visible stromal involvement on the slit lamp examination, corneal analysis based on confocal microscopy and optical coherence tomography revealed characteristic features of macular corneal dystrophy (MCD). The diagnosis of MCD was confirmed by CHST6 gene sequencing. The early corneal characteristic features of MCD, established based on the findings of this case report, include corneal astigmatism (not specific), diffuse corneal thinning without a pattern of corneal ectasia (specific), and characteristic features on confocal microscopy (specific), including multiple, dark, oriented striae at different corneal depths. CONCLUSIONS: The clinical examination should be complemented with corneal imaging techniques, such as confocal microscopy and optical coherence tomography. In patients suspected of corneal dystrophy, genetic testing plays an important role in establishing the final diagnosis.


Assuntos
Distrofias Hereditárias da Córnea , Opacidade da Córnea , Criança , Córnea , Distrofias Hereditárias da Córnea/diagnóstico , Distrofias Hereditárias da Córnea/genética , Humanos , Masculino , Microscopia Confocal , Tomografia de Coerência Óptica
7.
Chin J Dent Res ; 24(2): 113-118, 2021 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-34219444

RESUMO

OBJECTIVE: To compare the efficiency of a new sonic powered irrigation system named EDDY (VDW, Munich, Germany), passive ultrasonic irrigation (PUI) and conventional needle irrigation (CNI) in root canal sealer penetration. METHODS: A total of 45 mandibular premolars were instrumented up to size 30, 0.9 taper and randomly divided into three groups (n = 15) depending on the final irrigation activation technique: EDDY, PUI or CNI. After the final irrigation procedures, the root canals were obturated with labelled sealer mixed with 0.1% rhodamine B. Transverse sections at 3, 5 and 7 mm from the root apex were examined using confocal laser scanning microscopy. The maximum depth and total area and percentage of sealer penetration were measured using ImageJ analysis software (National Institutes of Health, Bethesda, MD, USA). RESULTS: In the EDDY group, the penetration depth was higher compared to the CNI group in the apical and middle sections and compared to the PUI group in the apical section (P ˂ 0.05). The penetration area in the EDDY group was higher compared to the CNI group in all sections and compared to the PUI group in the coronal section (P ˂ 0.05). The percentage of penetration was higher in the EDDY group compared to the CNI group in all sections and compared to the PUI group in the coronal section (P ˂ 0.05). CONCLUSION: In the present study, sealer penetration was superior in the EDDY group than the CNI group in the apical section. In the middle and coronal sections, sealer penetration was similar for the EDDY and PUI groups.


Assuntos
Materiais Restauradores do Canal Radicular , Irrigantes do Canal Radicular , Alemanha , Humanos , Microscopia Confocal , Preparo de Canal Radicular , Irrigação Terapêutica
8.
Int J Mol Sci ; 22(11)2021 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-34073457

RESUMO

To date, data on the presence of adenoviral receptors in fish are very limited. In the present work, we used mouse recombinant adeno-associated viral vectors (rAAV) with a calcium indicator of the latest generation GCaMP6m that are usually applied for the dorsal hippocampus of mice but were not previously used for gene delivery into fish brain. The aim of our work was to study the feasibility of transduction of rAAV in the mouse hippocampus into brain cells of juvenile chum salmon and subsequent determination of the phenotype of rAAV-labeled cells by confocal laser scanning microscopy (CLSM). Delivery of the gene in vivo was carried out by intracranial injection of a GCaMP6m-GFP-containing vector directly into the mesencephalic tegmentum region of juvenile (one-year-old) chum salmon, Oncorhynchus keta. AAV incorporation into brain cells of the juvenile chum salmon was assessed at 1 week after a single injection of the vector. AAV expression in various areas of the thalamus, pretectum, posterior-tuberal region, postcommissural region, medial and lateral regions of the tegmentum, and mesencephalic reticular formation of juvenile O. keta was evaluated using CLSM followed by immunohistochemical analysis of the localization of the neuron-specific calcium binding protein HuCD in combination with nuclear staining with DAPI. The results of the analysis showed partial colocalization of cells expressing GCaMP6m-GFP with red fluorescent HuCD protein. Thus, cells of the thalamus, posterior tuberal region, mesencephalic tegmentum, cells of the accessory visual system, mesencephalic reticular formation, hypothalamus, and postcommissural region of the mesencephalon of juvenile chum salmon expressing GCaMP6m-GFP were attributed to the neuron-specific line of chum salmon brain cells, which indicates the ability of hippocampal mammal rAAV to integrate into neurons of the central nervous system of fish with subsequent expression of viral proteins, which obviously indicates the neuronal expression of a mammalian adenoviral receptor homolog by juvenile chum salmon neurons.


Assuntos
Dependovirus , Vetores Genéticos , Neurônios , Oncorhynchus keta , Tegmento Mesencefálico , Transdução Genética , Animais , Camundongos , Microscopia Confocal , Neurônios/citologia , Neurônios/metabolismo , Oncorhynchus keta/genética , Oncorhynchus keta/metabolismo , Tegmento Mesencefálico/citologia , Tegmento Mesencefálico/metabolismo
9.
Int J Mol Sci ; 22(11)2021 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-34071318

RESUMO

Cathepsin K-mediated thyroglobulin proteolysis contributes to thyroid hormone (TH) liberation, while TH transporters like Mct8 and Mct10 ensure TH release from thyroid follicles into the blood circulation. Thus, thyroid stimulating hormone (TSH) released upon TH demand binds to TSH receptors of thyrocytes, where it triggers Gαq-mediated short-term effects like cathepsin-mediated thyroglobulin utilization, and Gαs-mediated long-term signaling responses like thyroglobulin biosynthesis and thyrocyte proliferation. As reported recently, mice lacking Mct8 and Mct10 on a cathepsin K-deficient background exhibit excessive thyroglobulin proteolysis hinting towards altered TSH receptor signaling. Indeed, a combination of canonical basolateral and non-canonical vesicular TSH receptor localization was observed in Ctsk-/-/Mct8-/y/Mct10-/- mice, which implies prolonged Gαs-mediated signaling since endo-lysosomal down-regulation of the TSH receptor was not detected. Inspection of single knockout genotypes revealed that the TSH receptor localizes basolaterally in Ctsk-/- and Mct8-/y mice, whereas its localization is restricted to vesicles in Mct10-/- thyrocytes. The additional lack of cathepsin K reverses this effect, because Ctsk-/-/Mct10-/- mice display TSH receptors basolaterally, thereby indicating that cathepsin K and Mct10 contribute to TSH receptor homeostasis by maintaining its canonical localization in thyrocytes. Moreover, Mct10-/- mice displayed reduced numbers of dead thyrocytes, while their thyroid gland morphology was comparable to wild-type controls. In contrast, Mct8-/y, Mct8-/y/Mct10-/-, and Ctsk-/-/Mct8-/y/Mct10-/- mice showed enlarged thyroid follicles and increased cell death, indicating that Mct8 deficiency results in altered thyroid morphology. We conclude that vesicular TSH receptor localization does not result in different thyroid tissue architecture; however, Mct10 deficiency possibly modulates TSH receptor signaling for regulating thyrocyte survival.


Assuntos
Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Receptores da Tireotropina/metabolismo , Células Epiteliais da Tireoide/metabolismo , Glândula Tireoide/metabolismo , Sistemas de Transporte de Aminoácidos Neutros/deficiência , Sistemas de Transporte de Aminoácidos Neutros/genética , Animais , Catepsina K/deficiência , Catepsina K/genética , Catepsina K/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Confocal , Tireoglobulina/metabolismo , Glândula Tireoide/citologia , Hormônios Tireóideos/metabolismo , Tireotropina/sangue , Tireotropina/metabolismo
10.
Int J Mol Sci ; 22(11)2021 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-34071406

RESUMO

Coralyne is a synthetic analog of berberine related to protoberberine-isoquinoline alkaloids. Isoquinoline derivatives and analogs are renowned as potent radiosensitizers with potential medical application. In the present study, we investigated the effect of coralyne on the cell death, cytoskeletal changes and cell cycle progression of irradiated A549 cells. A clonogenic assay revealed that coralyne pretreatment decreased the viability of A549 cells in a time- and dose-dependent manner. Moreover, exposure to coralyne and ionizing radiation (IR) markedly altered the filamentous actin cytoskeletal architecture and integrin-ß binding sites of A549 cells. Treatment with 1-25 µM coralyne in combination with 2 Gy of IR significantly reduced the percentage of cells in G2/M phase compared with 2 Gy IR alone. These results indicate that coralyne is a potent radiosensitizing agent that may find an application in medicine.


Assuntos
Alcaloides de Berberina/farmacologia , Inibidor de Quinase Dependente de Ciclina p21/genética , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Células A549 , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/efeitos da radiação , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Relação Dose-Resposta a Droga , Relação Dose-Resposta à Radiação , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos da radiação , Humanos , Microscopia Confocal , Radiação Ionizante , Radiossensibilizantes/farmacologia
11.
Molecules ; 26(10)2021 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-34070063

RESUMO

Amlodipine, a unique long-lasting calcium channel antagonist and antihypertensive drug, has weak fluorescence in aqueous solutions. In the current paper, we show that direct visualization of amlodipine in live cells is possible due to the enhanced emission in cellular environment. We examined the impact of pH, polarity and viscosity of the environment as well as protein binding on the spectral properties of amlodipine in vitro, and used quantum chemical calculations for assessing the mechanism of fluorescence quenching in aqueous solutions. The confocal fluorescence microscopy shows that the drug readily penetrates the plasma membrane and accumulates in the intracellular vesicles. Visible emission and photostability of amlodipine allow confocal time-lapse imaging and the drug uptake monitoring.


Assuntos
Anlodipino/farmacologia , Microscopia de Fluorescência , Anlodipino/química , Sobrevivência Celular/efeitos dos fármacos , Células HEK293 , Humanos , Indóis/metabolismo , Microscopia Confocal , Modelos Biológicos , Conformação Molecular , Soluções
12.
Vestn Oftalmol ; 137(3): 39-48, 2021.
Artigo em Russo | MEDLINE | ID: mdl-34156777

RESUMO

PURPOSE: To assess the state of corneal nerve fibers (CNF) based on quantitative parameters after laser correction of myopia using LASIK. MATERIAL AND METHODS: The study examined 141 eyes - 80 eyes with myopia and 61 emmetropic eyes. In 47 cases a microkeratome was used to form a corneal flap, and in 33 cases a femtosecond laser was used (LASIK and FemtoLASIK, respectively). Confocal microscopy was performed before, as well as 1, 3, and 6 months after correction. Objective analysis of CNF involved automatic calculation of the anisotropy (KΔL) and symmetry (Ksym) coefficients of CNF orientation in the Liner 1.2S software. RESULTS: Lower values of KΔL were revealed in myopia as compared to emmetropia. Regardless of the method of flap formation, no CNF could be detected in the central cornea 1 month after myopia correction. In all cases after FemtoLASIK and in 2 cases after LASIK, CNF in the central cornea were first visualized after 3 months, and after 6 months CNF could be detected in all cases. At a similar observation time, CNF were visualized both after LASIK and after FemtoLASIK in the superior and inferior peripheral portions of the cornea. Analysis of long-term postoperative changes in KΔL and Ksym revealed a decrease in the former and an increase in the latter. CONCLUSION: Evaluation of the state of CNF after corneal refractive correction should take into account the initial changes in CNF in myopia that are possibly associated with an increase in axial eye length. A more dynamic restoration of innervation was noted in cases where the flap was formed with the femtosecond laser, which may be explained by the fact that the mechanism of tissue separation when using this type of coherent radiation is gentler. Considering the changes in the quantitative indicators characterizing the state of CNF, the question of «completeness¼ of the restored CNF as a result of reinnervation remains open.


Assuntos
Ceratomileuse Assistida por Excimer Laser In Situ , Miopia , Córnea/diagnóstico por imagem , Córnea/cirurgia , Humanos , Ceratomileuse Assistida por Excimer Laser In Situ/efeitos adversos , Lasers de Excimer , Microscopia Confocal , Miopia/diagnóstico , Miopia/cirurgia , Fibras Nervosas
13.
Vestn Oftalmol ; 137(3): 58-67, 2021.
Artigo em Russo | MEDLINE | ID: mdl-34156779

RESUMO

Application of terahertz (THz) radiation in novel non-invasive biomedical technologies has recently received considerable attention. However, experimental data about the safety of exposure to THz radiation for biological objects (including eye structures in vivo) are limited. To our knowledge, the safety of THz reflectometry (frequency range of 0.30-0.40 THz) has not been closely examined in an animal model with subsequent morphological assessment of corneal tissues. PURPOSE: To assess the safety of pulsed THz radiation with various parameters (time, power, and frequency) for the cornea in a rabbit model. MATERIAL AND METHODS: The sample for the current study consisted of 18 Chinchilla rabbits (18 eyes). Corneal imaging and epithelial cell density before and after the exposure were evaluated using confocal laser scanning microscopy (CLSM). The histological study for objective assessment of the cornea state (day 1 and day 14) was performed after experiment termination. RESULTS: Single and multiple exposures of laser radiation at a frequency below 0.1 THz and power density below 30 nW/cm2 do not cause visible structural changes in any layers of the rabbit cornea. The results obtained in the long-term period showed insignificant reversible morphological changes only within the epithelium. CONCLUSION: The described parameters of terahertz and subterahertz radiation can be considered safe for assessing changes in corneal epithelium hydration level using non-invasive methods based on THz reflectometry.


Assuntos
Epitélio Corneano , Radiação Terahertz , Animais , Córnea , Microscopia Confocal , Coelhos
14.
BMC Ophthalmol ; 21(1): 261, 2021 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-34147078

RESUMO

BACKGROUND: To evaluate microstructural changes in the meibomian glands (MGs) in patients with active and inactive Graves' orbitopathy (GO), using in vivo confocal microscopy (IVCM), and to investigate the correlations between clinical and confocal findings. METHODS: Forty patients (80 eyes) with GO (34 eyes with active GO, 46 eyes with inactive GO), and 31 age- and sex-matched control participants (62 eyes) were enrolled consecutively. A researcher recorded the clinical activity score (CAS) for each patient. A complete ophthalmic examination was then performed, including external eye, ocular surface and MGs. IVCM of the MGs was performed to determine the MG acinar density (MAD), MG longest and shortest diameters (MALD and MASD), MG orifice area (MOA), MG acinar irregularity (MAI), meibum secretion reflectivity (MSR), acinar wall inhomogeneity (AWI), acinar periglandular interstices inhomogeneity (API), and severity of MG fibrosis (MF). RESULTS: All confocal microscopy assessments of MGs significantly differed among groups (all P = 0.000). Compared to controls, GO groups showed lower MOA (1985.82 ± 1325.30 µm2 in active GO and 2021.59 ± 1367.45 µm2 in inactive GO vs. 3896.63 ± 891.90 µm2 in controls, all P = 0.000) and MAD (87.21 ± 32.69 /mm2 in active GO and 80.72 ± 35.54 /mm2 in inactive GO vs. 114.69 ± 34.90 /mm2 in controls, P = 0.001 and 0.000, respectively); greater MALD (118.11 ± 30.23 µm in active GO and 120.58 ± 27.64 µm in inactive GO vs. 58.68 ± 20.28 µm in controls, all P = 0.000) and MASD (44.77 ± 19.16 µm in active GO and 46.02 ± 20.70 µm in inactive GO vs. 27.80 ± 9.90 µm in controls, all P = 0.000); and higher degrees of MAI, MSR, and MF (all P<0.05). Eyes with active GO had higher degrees of MAI (P = 0.015), AWI (P = 0.000), and API (P = 0.000), while eyes with inactive GO had higher degrees of MSR (P = 0.000) and MF (P = 0.017). In GO groups, AWI and API were positively correlated with CAS (r = 0.640, P = 0.000; r = 0.683, P = 0.000, respectively), and MF was negatively correlated with CAS (r = - 0.228, P = 0.042). CONCLUSIONS: IVCM effectively revealed microstructural changes of MGs in eyes with GO and provided strong in vivo evidence for the roles of obstruction and inflammation in the ocular surface disease process. Furthermore, it revealed discernible patterns of MG abnormalities in eyes with active GO and inactive GO, which are not easily distinguishable by typical clinical examinations.


Assuntos
Oftalmopatia de Graves , Fibrose , Oftalmopatia de Graves/diagnóstico por imagem , Oftalmopatia de Graves/patologia , Humanos , Glândulas Tarsais/diagnóstico por imagem , Glândulas Tarsais/patologia , Microscopia Confocal , Lágrimas
15.
Indian J Ophthalmol ; 69(7): 1730-1734, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34146016

RESUMO

Purpose: The aim of this study was to investigate the association between alterations in corneal subbasal nerve plexus and tactile corneal sensitivity in patients with Fuchs' endothelial corneal dystrophy (FECD). Methods: This retrospective, cross-sectional study included 24 (10 M/14 F) patients with FECD and 25 age- and sex-matched (10 M/15 F) healthy subjects as controls. Subjects with FECD were classified as having early (grades 1 and 2) and late (grades 3 and 4) disease. All subjects underwent central corneal tactile sensitivity measurements with the Cochet-Bonnet esthesiometer (Luneau Ophthalmologie, Chartres, France) and subbasal nerve density evaluation using in vivo confocal microscopy (IVCM). Association between corneal nerve plexus density and corneal sensitivity alterations were evaluated using the Mann-Whitney U test and the Spearman correlation test. Results: Compared to healthy subjects (mean age = 60.4 ± 7.5 years), patients with FECD (mean age = 60.6 ± 8.0 years) had worse central corneal sensitivity scores (5.9 ± 0.1 cm vs. 4.2 ± 0.8 cm; P < 0.001), reduced corneal nerve fibers (3.4 ± 1.3 nerves/frame vs. 5.0 ± 0.9 nerves/frame; P < 0.001) and lower corneal subbasal nerve plexus densities (2229.4 ± 364.3 µm/mm2 vs. 1901.6 ± 486.8 µm/mm2; P = 0.050). Patients with late stage FECD demonstrated lower subbasal nerve densities as compared to those with early disease (2204.3 ± 313.1 µm/mm2 (range = 1523-2552 µm/mm2); 1397.1 ± 227.4 µm/mm2 (range = 1120-1834 µm/mm2); P < 0.001). In the FECD group, subbasal nerve density was found to be directly correlated with corneal sensitivity scores (r = 0.457, P = 0.025). Conclusion: Progressive loss of the corneal subbasal nerve plexus appears to be a consistent feature of FECD. Reduction of the corneal nerve plexus parallels the decrease in corneal sensitivity in this patient population.


Assuntos
Distrofia Endotelial de Fuchs , Idoso , Córnea , Estudos Transversais , Humanos , Microscopia Confocal , Pessoa de Meia-Idade , Nervo Oftálmico , Estudos Prospectivos , Estudos Retrospectivos
16.
Nano Lett ; 21(12): 4950-4958, 2021 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-34125553

RESUMO

PIEZO1 ion channels are activated by mechanical stimuli, triggering intracellular chemical signals. Recent structural studies suggest that plasma membrane tension or local curvature changes modulate PIEZO1 channel gating and activation. However, whether PIEZO1 localization is governed by tension gradients or long-range mechanical perturbations across the cells is still unclear. Here, we probe the nanoscale localization of PIEZO1 on red blood cells (RBCs) at high resolution (∼30 nm), and we report for the first time the existence of submicrometric PIEZO1 clusters in native conditions. Upon interaction with Yoda1, an allosteric modulator, PIEZO1 clusters increase in abundance in regions of higher membrane tension and lower curvature. We further show that PIEZO1 ion channels interact with the spectrin cytoskeleton in both resting and activated states. Our results point toward a strong interplay between plasma membrane tension gradients, curvature, and cytoskeleton association of PIEZO1.


Assuntos
Canais Iônicos , Fenômenos Mecânicos , Membrana Celular/metabolismo , Citoesqueleto/metabolismo , Canais Iônicos/metabolismo , Mecanotransdução Celular , Microscopia Confocal
17.
Colloids Surf B Biointerfaces ; 205: 111881, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34062346

RESUMO

Nuclear breakdown was found to be the dominant route for DNA entry into the nucleus in actively dividing cells. The possibility that alternative routes contribute to DNA entry into the nucleus, however, cannot be ruled out. Here we address the process of lipofection by monitoring the localization of fluorescently-labelled DNA plasmids at the single-cell level by confocal imaging in living interphase cells. As test formulation we choose the cationic 3ß-[N-(N,N-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) and the zwitterionic helper lipid dioleoylphosphatidylethanolamine (DOPE) with plasmidic DNA pre-condensed by means of protamine. By exploiting the spectral shift of the fluorescent dye FM4-64 (N-(3-triethylammoniumpropyl)-4-(p-diethylaminophenylhexatrienyl)-pyridinium 2Br) we monitor the position of the nuclear envelope (NE), while concomitantly imaging the whole nucleus (by Hoechst) and the DNA (by Cy3 fluorophore) in a multi-channel 3D confocal imaging experiment. Reported results show that DNA clusters are typically associated with the NE membrane in the form of tubular invaginations spanning the nuclear environment, but not completely trapped within the NE invaginations, i.e. the DNA may use these NE regions as entry-points towards the nucleus. These observations pave the way to investigating the molecular details of the postulated processes for a better exploitation of gene-delivery vectors, particularly for applications in non-dividing cells.


Assuntos
Lipossomos , Membrana Nuclear , DNA , Microscopia Confocal , Transfecção
18.
Nucleic Acids Res ; 49(11): 6456-6473, 2021 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-34107032

RESUMO

RNA-protein interactions are central to all gene expression processes and contribute to a variety of human diseases. Therapeutic approaches targeting RNA-protein interactions have shown promising effects on some diseases that are previously regarded as 'incurable'. Here, we developed a fluorescent on-bead screening platform, RNA Pull-Down COnfocal NAnoscanning (RP-CONA), to identify RNA-protein interaction modulators in eukaryotic cell extracts. Using RP-CONA, we identified small molecules that disrupt the interaction between HuR, an inhibitor of brain-enriched miR-7 biogenesis, and the conserved terminal loop of pri-miR-7-1. Importantly, miR-7's primary target is an mRNA of α-synuclein, which contributes to the aetiology of Parkinson's disease. Our method identified a natural product quercetin as a molecule able to upregulate cellular miR-7 levels and downregulate the expression of α-synuclein. This opens up new therapeutic avenues towards treatment of Parkinson's disease as well as provides a novel methodology to search for modulators of RNA-protein interaction.


Assuntos
Proteína Semelhante a ELAV 1/antagonistas & inibidores , MicroRNAs/antagonistas & inibidores , Quercetina/farmacologia , alfa-Sinucleína/metabolismo , Avaliação Pré-Clínica de Medicamentos/métodos , Proteína Semelhante a ELAV 1/metabolismo , Células HEK293 , Células HeLa , Humanos , MicroRNAs/metabolismo , Microscopia Confocal , RNA Mensageiro/metabolismo , alfa-Sinucleína/genética
19.
Methods Mol Biol ; 2277: 157-173, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34080151

RESUMO

Mitochondria have complex ultrastructure which includes continuous subcompartments, such as matrix, intermembrane space, and two membranes, as well as focal structures, such as nucleoids, RNA granules, and mitoribosomes. Comprehensive studies of the spatial distribution of proteins and RNAs inside the mitochondria are necessary to understand organellar gene expression processes and macromolecule targeting pathways. Here we give examples of distribution analysis of mitochondrial proteins and transcripts by conventional microscopy and the super-resolution technique 3D STORM. We provide detailed protocols and discuss limitations of immunolabeling of mitochondrial proteins and newly synthesized mitochondrial RNAs by bromouridine incorporation and single-molecule RNA FISH in hepatocarcinoma cells.


Assuntos
Imuno-Histoquímica/métodos , Hibridização in Situ Fluorescente/métodos , Microscopia Confocal/métodos , Proteínas Mitocondriais/metabolismo , Bromouracila/análogos & derivados , Bromouracila/química , Células Hep G2 , Humanos , Processamento de Imagem Assistida por Computador/métodos , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Proteínas Mitocondriais/genética , RNA Mitocondrial/química , Imagem Individual de Molécula/métodos , Uridina/análogos & derivados , Uridina/química
20.
Methods Mol Biol ; 2277: 187-201, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34080153

RESUMO

Mitochondria, similar to living cells and organelles, have a negative membrane potential, which ranges between (-108) and (150) mV as compared to (-70) and (-90) mV of the plasma membrane. Therefore, permeable lipophilic cations tend to accumulate in the mitochondria. Those cations which exhibit fluorescence activity after accumulation into energized systems are widely used to decipher changes in membrane potential by imaging techniques. Here we describe the use of two different dyes for labeling mitochondrial membrane potential (Δψm) in live cells. One is the lipophilic cation 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazol-carbocyanine iodide (JC-1), which alters reversibly its color from green (J-monomer, at its low concentration in the cytosol) to red (J-aggregates, at its high concentration in active mitochondria) with increasing mitochondrial membrane potential (Δψm). The other is MitoTracker® Orange, a mitochondrion-selective probe which passively diffuses across the plasma membrane and accumulates in active mitochondria depending on their Δψm. We show that in addition to changes in Δψm, these specific dyes can be used to follow alterations in mitochondrial distribution and mitochondrial network connectivity. We suggest that JC-1 is a preferable probe to compare between different cell types and cell state, as a red to green ratio of fluorescence intensities is used for analysis. This ratio depends only on the mitochondrial membrane potential and not on other cellular and/or mitochondrial-dependent or independent factors that may alter, for example, due to treatment or disease state. However, in cells labeled either with green or red fluorescence protein, JC-1 cannot be used. Therefore, other dyes are preferable. We demonstrate various applications of JC-1 and MitoTracker Orange staining to study mitochondrial abnormalities in different cell types derived from schizophrenia patients and healthy subjects.


Assuntos
Processamento de Imagem Assistida por Computador/métodos , Potencial da Membrana Mitocondrial , Mitocôndrias/metabolismo , Esquizofrenia/metabolismo , Benzimidazóis/química , Carbocianinas/química , Técnicas de Cultura de Células , Fibroblastos/metabolismo , Fibroblastos/patologia , Corantes Fluorescentes/química , Humanos , Microscopia Confocal/instrumentação , Microscopia Confocal/métodos , Mitocôndrias/patologia , Estudo de Prova de Conceito , Esquizofrenia/patologia , Xantenos/química
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